The genome sequence of the Large Red Damselfly Pyrrhosoma nymphula (Sulzer, 1776)

We present a genome assembly from an individual male Pyrrhosoma nymphula (the Large Red Damselfly; Arthropoda; Insecta; Odonata; Coenagrionidae). The genome sequence is 2,117.2 megabases in span. Most of the assembly is scaffolded into 14 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 16.78 kilobases in length.


Background
The Large Red Damselfly, Pyrrhosoma nymphula is, as its common name would suggest, a large, red damselfly in the order Odonata.Black markings on the abdomen occur in three forms in the female and are less numerous in the male.Pyrrhosoma nymphula has a hind wing length of 19-24 mm, an abdominal length of 25-29 mm and a mean weight of 40 g for females and 37 g for males (Brooks, 2007) .
Pyrrhosoma nymphula is a species of the West Palaearctic region (Guan et al., 2013) and it is widely distributed throughout the United Kingdom and Ireland, where it breeds in still or slow-moving water, such as ponds, bogs, ditches and canals (Brooks, 2007).Adults can also be found feeding on smaller insects in sunlit areas in woodlands and along hedgerows.
Across Europe, Pyrrhosoma nymphula are some of the earliest damselflies to emerge in spring (Guan et al., 2013) and in the United Kingdom and Ireland are on the wing from late April to July and occasionally into September (Brooks, 2007).After emergence, the males take about 12 days to mature and females 16 days (Brooks, 2007).Males defend a territory, consisting of a perch and a small area of airspace around it, to watch for females, and the resident males win the vast majority of territorial disputes regardless of their size (Gribbin & Thompson, 1991).Eggs are deposited into submerged vegetation while the pair flies in tandem (Brooks, 2007).
The Large Red Damselfly's lifecycle usually takes two years but, if food is scarce, it may take three years for the aquatic carnivorous larvae to reach maturity (Brooks, 2007).Like the adults, the final instar larvae are also territorial, with occupants observed to defeat intruders on 72% of occasions (Harvey & Corbet, 1986).
We present a chromosomal-level genome sequence for a male Pyrrhosoma nymphula, based on one specimen collected from Wytham Woods, Oxfordshire, UK as part of the Darwin Tree of Life Project.

Genome sequence report
The genome was sequenced from adult male Pyrrhosoma nymphula (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.76,.A total of 35-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 51 missing joins or mis-joins, reducing the scaffold number by 11.26%, and increasing the scaffold N50 by 0.62%. The final assembly has a total length of 2,117.2Mb in 322 sequence scaffolds with a scaffold N50 of 156.2 Mb (Table 1).The snail plot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (99.77%) of the assembly sequence was assigned to 14 chromosomal-level scaffolds, representing 13 autosomes and the X sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).The sample is that of an XO male.The order and orientation of contigs along Chromosome 13 from 3.5 Mb to 21 Mb is uncertain.While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.
The estimated Quality Value (QV) of the final assembly is 62.2 with k-mer completeness of 100.0%, and the assembly has a BUSCO v5.4.3 completeness of 96.9% (single = 96.4%,duplicated = 0.5%), using the insecta_odb10 reference set (n = 1,367).sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up.In sample preparation, the ioPyrNymp1 sample was weighed and dissected on dry ice (Jay et al., 2023) and tissue from the thorax was homogenised using a PowerMasher II tissue disruptor (Denton et al., 2023a).
HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol (Oatley et al., 2023).The DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 31 (Bates et al., 2023).Sheared DNA was purified by solid-phase reversible immobilisation (Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA.The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit.Fragment size distribution was evaluated by running the sample on the FemtoPulse system.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on a Pacific Biosciences Revio instrument.Hi-C data were also generated from head tissue of ioPyrNymp1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.The sanger-tol/blobtoolkit pipeline is a Nextflow port of the previous Snakemake Blobtoolkit pipeline (Challis et al., 2020).It aligns the PacBio reads with SAMtools and mini-map2 (Li, 2018) and generates coverage tracks for regions of fixed size.In parallel, it queries the GoaT database (Challis et al., 2023) to identify all matching BUSCO lineages to run BUSCO (Manni et al., 2021).For the three domain-level BUSCO lineage, the pipeline aligns the BUSCO genes to the Uniprot Reference Proteomes database (Bateman et al., 2023) with DIAMOND (Buchfink et al., 2021) blastp.The genome is also split into chunks according to the density of the BUSCO genes from the closest taxonomically lineage, and each chunk is aligned to the Uniprot Reference Proteomes database with DIAMOND blastx.Genome sequences that have no hit are then chunked with seqtk and aligned to the NT database with blastn (Altschul et al., 1990).All those outputs are combined with the blobtools suite into a blobdir for visualisation.
All three pipelines were developed using the nf-core tooling (Ewels et al., 2020)

Software tool Version
Wellcome Sanger Institute -Legal and Governance The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is subject to the 'Darwin Tree of Life Project Sampling Code of Practice', which can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.
Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material

Strengths
The paper focuses on the technical aspects of the genome assembly, with relatively little discussion on the biological implications of the genomic data.There is limited discussion on how this genome sequence compares to other damselflies.Comparative genomics could provide more context on the uniqueness and conservation of genomic features.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Molecular cytogenetics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Yesenia Margarita Vega-Sánchez
Instituto de Investigaciones en Ecosistemas y Sustentabilidad, Universidad Nacional Autónoma de México (UNAM), Morelia, Mexico The Data Note describes a chromosome-level genome assembly of Pyrrhosoma nymphula, an insect species of the Order Odonata.
In general, the manuscript is well developed; except for some paragraphs that seem redundant, for example the first and last paragraphs of the Background.The methods are concise and sufficient for the repeatability of the study.The results are clear, however, in my opinion, there are some images that are not necessary (e.g.Fig. 3 and Fig. 4).I have no additional comments.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
2 National University of Singapore, Singapore, Singapore This data note describes an assembly for the Large Red Damselfly Pyrrhosoma nymphula.
Damselflies are ancient insects with striking ecological, behavioural, and morphological diversity, but with relatively limited genomic resources to investigate the molecular basis, evolution, and threats to this diversity.I thus think that this work provides an important resource for future research.The data is of high quality and the note is generally well written.I have a few suggestions to improve clarity and provide biological context to the manuscript.
1.The Background section briefly mentions the occurrence of sexual dimorphism in colour and female-limited colour morphs in P. nymphula: "Black markings on the abdomen occur in three forms in the female and are less numerous in the male".
I think this brief mention could be expanded to highlight one of the potential uses of this assembly.P. nymphula belongs to Coenagrionidae, a damselfly family in which females often display colour polymorphisms, but in which males are typically monomorphic (see for example Fincke et al. 2005).As the authors mention, there are three female morphs in P. nymphula, which vary in the extent of black abdomen markings and thus in the extent of their resemblance to males (Askew 2004).
This genome assembly creates the opportunity to investigate the genetic basis of this sex-specific colour variation.There have been a few recent studies looking into the genetics of female-limited polymorphisms in coenagrionid damselflies (Takahashi et al. 2019, Willink et al. 2024), but I think P. nymphula is really interesting, because female morphs in this species show a gradient of phenotypic differentiation from males.One morph (fulvipes) has a mostly red abdomen, similar to males.Another morph (melanotum) has a mostly black abdomen, and the third morph (intermedia or typica) has some red and some black markings.This assembly provides a great resource to investigate the genetics of this polymorphism, perhaps in the larger context of the evolution of sexual dimorphism.
2. Another potential application for this assembly could be for biogeographic and speciation research.I'm not so familiar with this literature, but I understand there are two lineages that diverged from the more widespread P. nymphula lineage, one in Northern Africa, and one in costal Greece.The lineage in costal Greece, currently considered a separate species (P.elisabethae), is critically endangered (see IUCN redlist).There is some older and more recent research on this topic that might be worth revising (e.g.Kalkman and Lopau 2006, Guan et al. 2013, Simonsen et al. 2024).I imagine that this assembly can help shed light into the interesting biogeographic history and evolutionary relationships within this clade.
3. I understand this is the most common sex determination system in Odonata, but I wonder how the authors determined that their male sample is XO.

Fig 3:
this is explained in the text, but the x and y axes should also be described in the figure legend as well."gc" and "ERR6688447_cov" are not intuitive axes titles.
5. Perhaps the legend of Figure 4 use "scaffolds" instead of "sequence".Otherwise, it's ambiguous whether "sequence" refers to raw reads or assembled scaffolds.
6. Fig. 4: Why is the background different in chromosome 6?Are there fewer HiC contacts?Or may be it just looks so against the white background.I think clarifying this is required to make the article scientifically sound.
7. I found that the "Evaluation of final assembly" section was difficult to follow.I think that revising this section is required to make the article scientifically sound.Specifically:  et al., 2018)." In the following sentence "It also provides statistics about the assembly...", clarify what you are referring to with "It".For example, "The sanger-tol/genomenote also provides statistics about the assembly..." or "HiGlass also provides statistics about the assembly...", whichever is correct.
Also in this sentence, I think the citation should be placed after "report".
The next paragraph describes the sanger-tol/blobtoolkit, but it is unclear to me what are the results of this evaluation step.The paragraph mentions coverage tracks for regions of a fixed size, BUSCO analyses for domain-level lineages, alignments to the Uniprot Reference Proteomes and to the NT database, which are all combined into a "blobdir for visualisation".Should this visualization be on the paper or available through a link?
The snail plot on Fig. 2 shows other, perhaps more basic assembly statistics.Considering that this sanger-tol/blobtoolkit pipeline is relatively new, it might be useful to mention in not so technical language how the pipeline contributes to the evaluation of the assembly, in addition of presenting its results.
8.There is an extra space before "." at the end of the first paragraph, in the "Background" section.

Figure 2 .
Figure 2. Genome assembly of Pyrrhosoma nymphula, ioPyrNymp1.1:metrics.The BlobToolKit snail plot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 2,117,243,717 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (205,116,909 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (156,229,410 and 129,911,903 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the insecta_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Pyrrhosoma_nymphula/dataset/GCA_963573305.1/snail.

Figure 3 .
Figure 3. Genome assembly of Pyrrhosoma nymphula, ioPyrNymp1.1:BlobToolKit GC-coverage plot.Sequences are coloured by phylum.Circles are sized in proportion to sequence length.Histograms show the distribution of sequence length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Pyrrhosoma_nymphula/dataset/GCA_963573305.1/blob.

Figure 4 .
Figure 4. Genome assembly of Pyrrhosoma nymphula ioPyrNymp1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Pyrrhosoma_nymphula/dataset/GCA_963573305.1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Pyrrhosoma nymphula ioPyrNymp1.1:Hi-C contact map of the ioPyrNymp1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=VNQ6n2laREeQQMNmjyDBGw.

Table 1 . Genome data for Pyrrhosoma nymphula, ioPyrNymp1.1. Project accession data
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of core procedures:

Table 3
contains a list of relevant software tool versions and sources.

Table 3 . Software tools: versions and sources.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

"
The sanger-tol/genomenote pipeline transforms the Hi-C alignments into a contact map with BEDTools (Quinlan & Hall, 2010) and the Cooler tool suite (Abdennur & Mirny, 2020), which is then visualised with HiGlass(Kerpedjiev et al., 2018)."Thislong sentence would be easier to read if split into two.One suggestion, assuming the meaning is preserved, could be: "The sanger-tol/genomenote pipeline transforms the Hi-C alignments into a contact map with BEDTools (Quinlan & Hall, 2010) and with the Cooler tool suite (Abdennur & Mirny, 2020).The contact map was then visualised with HiGlass (Kerpedjiev

Is the rationale for creating the dataset(s) clearly described? Partly Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Partly Competing Interests:
Publisher Full Text 5. Simonsen T, Djernaes M, Nielsen O, Olsen K: Increased geographic sampling suggests incomplete lineage sorting and recent introgression between Pyrrhosoma nymphula (Sulzer, 1776) and P. elisabethae Schmidt, 1948 in the Western Palearctic.International Journal of Odonatology.2024; 27: 37-46 Publisher Full Text 6. Takahashi M, Takahashi Y, Kawata M: Candidate genes associated with color morphs of femalelimited polymorphisms of the damselfly Ischnura senegalensis.Heredity (Edinb).2019; 122 (1): 81-92 PubMed Abstract | Publisher Full Text 7. Willink B, Tunström K, Nilén S, Chikhi R, et al.: The genomics and evolution of inter-sexual mimicry and female-limited polymorphisms in damselflies.Nat Ecol Evol.2024; 8 (1): 83-97 PubMed Abstract | Publisher Full Text No competing interests were disclosed.